Dihydrofolate reductase, or DHFR, is an
enzyme
An enzyme () is a protein that acts as a biological catalyst by accelerating chemical reactions. The molecules upon which enzymes may act are called substrate (chemistry), substrates, and the enzyme converts the substrates into different mol ...
that reduces
dihydrofolic acid to
tetrahydrofolic acid, using
NADPH
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require N ...
as an
electron donor
In chemistry, an electron donor is a chemical entity that transfers electrons to another compound. It is a reducing agent that, by virtue of its donating electrons, is itself oxidized in the process. An obsolete definition equated an electron dono ...
, which can be converted to the kinds of tetrahydrofolate
cofactors used in one-carbon transfer chemistry. In humans, the DHFR enzyme is encoded by the ''DHFR''
gene
In biology, the word gene has two meanings. The Mendelian gene is a basic unit of heredity. The molecular gene is a sequence of nucleotides in DNA that is transcribed to produce a functional RNA. There are two types of molecular genes: protei ...
.
It is found in the q14.1 region of chromosome 5.
There are two structural classes of DHFR, evolutionarily unrelated to each other. The former is usually just called DHFR and is found in bacterial chromosomes and animals. In bacteria, however, antibiotic pressure has caused this class to evolve different patterns of binding diaminoheterocyclic molecules, leading to many "types" named under this class, while
mammal
A mammal () is a vertebrate animal of the Class (biology), class Mammalia (). Mammals are characterised by the presence of milk-producing mammary glands for feeding their young, a broad neocortex region of the brain, fur or hair, and three ...
ian ones remain highly similar.
The latter (type II), represented by the plastid-encoded R67, is a tiny enzyme that works by forming a homotetramer.
Function
Dihydrofolate reductase converts
dihydrofolate into
tetrahydrofolate, a proton shuttle required for the de novo synthesis of
purines
Purine is a heterocyclic compound, heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which inc ...
,
thymidylic acid, and certain
amino acid
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although over 500 amino acids exist in nature, by far the most important are the 22 α-amino acids incorporated into proteins. Only these 22 a ...
s. While the functional dihydrofolate reductase gene has been mapped to chromosome 5, multiple intronless processed pseudogenes or dihydrofolate reductase-like genes have been identified on separate chromosomes.
Image:DHFR rxn.svg, Reaction catalyzed by DHFR.
Image:THFsynthesispathway.png, Tetrahydrofolate synthesis pathway.
Found in all organisms, DHFR has a critical role in regulating the amount of tetrahydrofolate in the cell. Tetrahydrofolate and its derivatives are essential for
purine
Purine is a heterocyclic aromatic organic compound that consists of two rings (pyrimidine and imidazole) fused together. It is water-soluble. Purine also gives its name to the wider class of molecules, purines, which include substituted puri ...
and
thymidylate synthesis, which are important for cell proliferation and cell growth.
DHFR plays a central role in the synthesis of
nucleic acid
Nucleic acids are large biomolecules that are crucial in all cells and viruses. They are composed of nucleotides, which are the monomer components: a pentose, 5-carbon sugar, a phosphate group and a nitrogenous base. The two main classes of nuclei ...
precursors, and it has been shown that mutant cells that completely lack DHFR require glycine, a purine, and thymidine to grow.
DHFR has also been demonstrated as an enzyme involved in the salvage of
tetrahydrobiopterin
Tetrahydrobiopterin (BH4, THB), also known as sapropterin ( INN), is a cofactor of the three aromatic amino acid hydroxylase enzymes, used in the metabolism of amino acid phenylalanine and in the biosynthesis of the neurotransmitters serotoni ...
from
dihydrobiopterin.
Structure
A central eight-stranded
beta-pleated sheet makes up the main feature of the
polypeptide
Peptides are short chains of amino acids linked by peptide bonds. A polypeptide is a longer, continuous, unbranched peptide chain. Polypeptides that have a molecular mass of 10,000 Da or more are called proteins. Chains of fewer than twenty ...
backbone folding of DHFR.
Seven of these strands are parallel and the eighth runs antiparallel. Four
alpha helices
An alpha helix (or α-helix) is a sequence of amino acids in a protein that are twisted into a coil (a helix).
The alpha helix is the most common structural arrangement in the secondary structure of proteins. It is also the most extreme type of l ...
connect successive beta strands.
Residues 9–24 are termed "Met20" or "loop 1" and, along with other loops, are part of the major subdomain that surround the
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
.
The
active site
In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the ''binding s ...
is situated in the
N-terminal
The N-terminus (also known as the amino-terminus, NH2-terminus, N-terminal end or amine-terminus) is the start of a protein or polypeptide, referring to the free amine group (-NH2) located at the end of a polypeptide. Within a peptide, the amin ...
half of the sequence, which includes a
conserved Pro
Pro is an abbreviation meaning "professional".
Pro, PRO or variants thereof might also refer to:
People
* Miguel Pro (1891–1927), Mexican priest
* Pro Hart (1928–2006), Australian painter
* Mlungisi Mdluli (born 1980), South African ret ...
-
Trp dipeptide; the
tryptophan
Tryptophan (symbol Trp or W)
is an α-amino acid that is used in the biosynthesis of proteins. Tryptophan contains an α-amino group, an α-carboxylic acid group, and a side chain indole, making it a polar molecule with a non-polar aromat ...
has been shown to be involved in the binding of
substrate
Substrate may refer to:
Physical layers
*Substrate (biology), the natural environment in which an organism lives, or the surface or medium on which an organism grows or is attached
** Substrate (aquatic environment), the earthy material that exi ...
by the enzyme.
Mechanism
General mechanism

DHFR catalyzes the transfer of a hydride from
NADPH
Nicotinamide adenine dinucleotide phosphate, abbreviated NADP or, in older notation, TPN (triphosphopyridine nucleotide), is a cofactor used in anabolic reactions, such as the Calvin cycle and lipid and nucleic acid syntheses, which require N ...
to
dihydrofolate with an accompanying protonation to produce
tetrahydrofolate.
In the end, dihydrofolate is reduced to tetrahydrofolate and NADPH is oxidized to
NADP+. The high flexibility of Met20 and other loops near the active site play a role in promoting the release of the product, tetrahydrofolate. In particular the Met20 loop helps stabilize the nicotinamide ring of the NADPH to promote the transfer of the hydride from NADPH to dihydrofolate.
The mechanism of this enzyme is stepwise and steady-state random. Specifically, the catalytic reaction begins with the NADPH and the substrate attaching to the binding site of the enzyme, followed by the protonation and the hydride transfer from the cofactor NADPH to the substrate. However, two latter steps do not take place simultaneously in a same transition state.
In a study using computational and experimental approaches, Liu ''et al'' conclude that the protonation step precedes the hydride transfer.
DHFR's enzymatic mechanism is shown to be pH dependent, particularly the hydride transfer step, since pH changes are shown to have remarkable influence on the electrostatics of the active site and the ionization state of its residues.
The acidity of the targeted nitrogen on the substrate is important in the binding of the substrate to the enzyme's binding site which is proved to be hydrophobic even though it has direct contact to water.
Asp27 is the only charged hydrophilic residue in the binding site, and neutralization of the charge on Asp27 may alter the pKa of the enzyme. Asp27 plays a critical role in the catalytic mechanism by helping with protonation of the substrate and restraining the substrate in the conformation favorable for the hydride transfer.
The protonation step is shown to be associated with enol tautomerization even though this conversion is not considered favorable for the proton donation.
A water molecule is proved to be involved in the protonation step.
Entry of the water molecule to the active site of the enzyme is facilitated by the Met20 loop.
Conformational changes of DHFR

The catalytic cycle of the reaction catalyzed by DHFR incorporates five important intermediate: holoenzyme (E:NADPH), Michaelis complex (E:NADPH:DHF), ternary product complex (E:NADP
+:THF), tetrahydrofolate binary complex (E:THF), and THF‚NADPH complex (E:NADPH:THF). The product (THF) dissociation step from E:NADPH:THF to E:NADPH is the rate determining step during steady-state turnover.
Conformational changes are critical in DHFR's catalytic mechanism. The Met20 loop of DHFR is able to open, close or occlude the active site.
Correspondingly, three different conformations classified as the opened, closed and occluded states are assigned to Met20. In addition, an extra distorted conformation of Met20 was defined due to its indistinct characterization results.
The Met20 loop is observed in its occluded conformation in the three product ligating intermediates, where the nicotinamide ring is occluded from the active site. This conformational feature accounts for the fact that the substitution of NADP
+ by NADPH is prior to product dissociation. Thus, the next round of reaction can occur upon the binding of substrate.
R67 DHFR
Due to its unique structure and catalytic features, R67 DHFR is widely studied. R67 DHFR is a type II R-plasmid-encoded DHFR without geneticay or structural relation to the E. coli chromosomal DHFR. It is a homotetramer that possesses the 222 symmetry with a single active site pore that is exposed to solvent. This symmetry of active site results in the different binding mode of the enzyme: It can bind with two dihydrofolate (DHF) molecules with positive cooperativity or two NADPH molecules with negative cooperativity, or one substrate plus one, but only the latter one has the catalytical activity. Compare with E. coli chromosomal DHFR, it has higher K
m in binding dihydrofolate (DHF) and NADPH. The much lower catalytical kinetics show that hydride transfer is the rate determine step rather than product (THF) release.
In the R67 DHFR structure, the homotetramer forms an active site pore. In the catalytical process, DHF and NADPH enters into the pore from opposite position. The π-π stacking interaction between NADPH's nicotinamide ring and DHF's pteridine ring tightly connect two reactants in the active site. However, the flexibility of p-aminobenzoylglutamate tail of DHF was observed upon binding which can promote the formation of the
transition state
In chemistry, the transition state of a chemical reaction is a particular configuration along the reaction coordinate. It is defined as the state corresponding to the highest potential energy along this reaction coordinate. It is often marked w ...
.
File:Reaction Kinetics comparison between EcDHFR and R67 DHFR.png, Reaction Kinetics comparison between ''E. coli'' DHFR (EcDHFR) and R67 DHFR
File:Structure difference of substrate binding in E. coli and R67 DHFR.png, Structure difference of substrate binding in EcDHFR and R67 DHFR
Clinical significance
DHFR mutations cause
dihydrofolate reductase deficiency, a rare autosomal recessive inborn error of folate metabolism that results in
megaloblastic anemia
Megaloblastic anemia is a type of macrocytic anemia. An anemia is a red blood cell defect that can lead to an undersupply of oxygen. Megaloblastic anemia results from inhibition of DNA replication, DNA synthesis during red blood cell production. ...
,
pancytopenia
Pancytopenia is a medical condition in which there is significant reduction in the number of almost all blood cells (red blood cells, white blood cells, platelets, monocytes, lymphocytes, etc.).
If only two parameters from the complete blood cou ...
and severe
cerebral folate deficiency. These issues can be overcome by supplementation with a reduced form of folate, usually
folinic acid
Folinic acid, also known as leucovorin, is a medication used to decrease the toxic effects of methotrexate and pyrimethamine. It is also used in combination with 5-fluorouracil to treat colorectal cancer and pancreatic cancer, may be used to ...
.
Therapeutic applications
DHFR is an attractive pharmaceutical target for inhibition due to its pivotal role in DNA precursor (
thymine
Thymine () (symbol T or Thy) is one of the four nucleotide bases in the nucleic acid of DNA that are represented by the letters G–C–A–T. The others are adenine, guanine, and cytosine. Thymine is also known as 5-methyluracil, a pyrimidine ...
) synthesis.
Trimethoprim
Trimethoprim (TMP) is an antibiotic used mainly in the treatment of bladder infections. Other uses include for middle ear infections and travelers' diarrhea. With sulfamethoxazole or dapsone it may be used for ''Pneumocystis'' pneumonia ...
, an
antibiotic
An antibiotic is a type of antimicrobial substance active against bacteria. It is the most important type of antibacterial agent for fighting pathogenic bacteria, bacterial infections, and antibiotic medications are widely used in the therapy ...
, inhibits bacterial DHFR while
methotrexate
Methotrexate, formerly known as amethopterin, is a chemotherapy agent and immunosuppressive drug, immune-system suppressant. It is used to treat cancer, autoimmune diseases, and ectopic pregnancy, ectopic pregnancies. Types of cancers it is u ...
, a
chemotherapy
Chemotherapy (often abbreviated chemo, sometimes CTX and CTx) is the type of cancer treatment that uses one or more anti-cancer drugs (list of chemotherapeutic agents, chemotherapeutic agents or alkylating agents) in a standard chemotherapy re ...
agent, inhibits mammalian DHFR. However,
resistance has developed against some drugs, as a result of mutational changes in DHFR itself.
Cancer
DHFR is responsible for the levels of tetrahydrofolate in a cell, and the inhibition of DHFR can limit the growth and proliferation of cells that are characteristic of cancer and bacterial infections.
Methotrexate
Methotrexate, formerly known as amethopterin, is a chemotherapy agent and immunosuppressive drug, immune-system suppressant. It is used to treat cancer, autoimmune diseases, and ectopic pregnancy, ectopic pregnancies. Types of cancers it is u ...
, a
competitive inhibitor
Competitive inhibition is interruption of a chemical pathway owing to one chemical substance inhibiting the effect of another by competing with it for binding or bonding. Any metabolic or chemical messenger system can potentially be affected b ...
of DHFR, is one such anticancer drug that inhibits DHFR.
Folate
Folate, also known as vitamin B9 and folacin, is one of the B vitamins. Manufactured folic acid, which is converted into folate by the body, is used as a dietary supplement and in food fortification as it is more stable during processing and ...
is necessary for growth,
and the pathway of the metabolism of folate is a target in developing treatments for cancer. DHFR is one such target. A regimen of
fluorouracil
Fluorouracil (5-FU, 5-fluorouracil), sold under the brand name Adrucil among others, is a cytotoxic chemotherapy medication used to treat cancer. By intravenous injection it is used for treatment of colorectal cancer, oesophageal cancer, stoma ...
,
doxorubicin
Doxorubicin, sold under the brand name Adriamycin among others, is a chemotherapy medication used to treat cancer. This includes breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphocytic leukemia. It is often used toge ...
, and methotrexate was shown to prolong survival in patients with advanced gastric cancer.
Further studies into inhibitors of DHFR can lead to more ways to treat cancer.
Infection
Bacteria also need DHFR to grow and multiply and hence inhibitors selective for bacterial DHFR have found application as antibacterial agents.
Trimethoprim has shown to have activity against a variety of
Gram-positive
In bacteriology, gram-positive bacteria are bacteria that give a positive result in the Gram stain test, which is traditionally used to quickly classify bacteria into two broad categories according to their type of cell wall.
The Gram stain is ...
bacterial pathogens.
However, resistance to trimethoprim and other drugs aimed at DHFR can arise due to a variety of mechanisms, limiting the success of their therapeutical uses.
Resistance can arise from DHFR gene amplification,
mutations
In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosi ...
in DHFR, decrease in the uptake of the drugs, among others. Regardless, trimethoprim and
sulfamethoxazole
Sulfamethoxazole (SMZ or SMX) is an antibiotic. It is used for bacterial infections such as urinary tract infections, bronchitis, and prostatitis and is effective against both gram negative and positive bacteria such as ''Escherichia coli' ...
in combination has been used as an antibacterial agent for decades.
Pyrimethamine
Pyrimethamine, sold under the brand name Daraprim among others, is a medication used with leucovorin (leucovorin is used to decrease side effects of pyrimethamine; it does not have intrinsic anti-parasitic activity) to treat the parasitic disea ...
is a widely used
antiprotozoal agent.
Other classes of compounds that target DHFR in general, and bacterial DHFRs in particular, belong to the classes such as diaminopteridines, diaminotriazines, diaminopyrroloquinazolines, stilbenes, chalcones, deoxybenzoins, diaminoquinazolines, diaminopyrroloquinazolines, to name but a few.
Potential anthrax treatment

Dihydrofolate reductase from ''
Bacillus anthracis
''Bacillus anthracis'' is a gram-positive and rod-shaped bacterium that causes anthrax, a deadly disease to livestock and, occasionally, to humans. It is the only permanent (obligate) pathogen within the genus ''Bacillus''. Its infection is a ty ...
'' (BaDHFR) is a validated drug target in the treatment of the infectious disease, anthrax. BaDHFR is less sensitive to
trimethoprim
Trimethoprim (TMP) is an antibiotic used mainly in the treatment of bladder infections. Other uses include for middle ear infections and travelers' diarrhea. With sulfamethoxazole or dapsone it may be used for ''Pneumocystis'' pneumonia ...
analogs than is dihydrofolate reductase from other species such as ''
Escherichia coli
''Escherichia coli'' ( )Wells, J. C. (2000) Longman Pronunciation Dictionary. Harlow ngland Pearson Education Ltd. is a gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus '' Escherichia'' that is commonly fo ...
'', ''
Staphylococcus aureus
''Staphylococcus aureus'' is a Gram-positive spherically shaped bacterium, a member of the Bacillota, and is a usual member of the microbiota of the body, frequently found in the upper respiratory tract and on the skin. It is often posi ...
'', and ''
Streptococcus pneumoniae
''Streptococcus pneumoniae'', or pneumococcus, is a Gram-positive, spherical bacteria, hemolysis (microbiology), alpha-hemolytic member of the genus ''Streptococcus''. ''S. pneumoniae'' cells are usually found in pairs (diplococci) and do not f ...
''. A structural alignment of dihydrofolate reductase from all four species shows that only BaDHFR has the combination
phenylalanine
Phenylalanine (symbol Phe or F) is an essential α-amino acid with the chemical formula, formula . It can be viewed as a benzyl group substituent, substituted for the methyl group of alanine, or a phenyl group in place of a terminal hydrogen of ...
and
tyrosine
-Tyrosine or tyrosine (symbol Tyr or Y) or 4-hydroxyphenylalanine is one of the 20 standard amino acids that are used by cells to synthesize proteins. It is a conditionally essential amino acid with a polar side group. The word "tyrosine" is ...
in positions 96 and 102, respectively.
BaDHFR's resistance to
trimethoprim
Trimethoprim (TMP) is an antibiotic used mainly in the treatment of bladder infections. Other uses include for middle ear infections and travelers' diarrhea. With sulfamethoxazole or dapsone it may be used for ''Pneumocystis'' pneumonia ...
analogs is due to these two residues (F96 and Y102), which also confer improved kinetics and catalytic efficiency.
Current research uses active site mutants in BaDHFR to guide lead optimization for new antifolate inhibitors.
As a research tool
DHFR has been used as a tool to detect
protein–protein interactions in a
protein-fragment complementation assay (PCA), using a split-protein approach.
DHFR-lacking
CHO cells are the most commonly used
cell line
An immortalised cell line is a population of cells from a multicellular organism that would normally not proliferate indefinitely but, due to mutation, have evaded normal cellular senescence and instead can keep undergoing division. The cells ...
for the production of recombinant proteins. These cells are
transfected with a
plasmid
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria and ...
carrying the ''dhfr'' gene and the gene for the recombinant protein in a single
expression system, and then subjected to
selective conditions in thymidine-lacking
medium
Medium may refer to:
Aircraft
*Medium bomber, a class of warplane
* Tecma Medium, a French hang glider design Arts, entertainment, and media Films
* ''The Medium'' (1921 film), a German silent film
* ''The Medium'' (1951 film), a film vers ...
. Only the cells with the exogenous DHFR gene along with the gene of interest survive. Supplementation of this medium with methotrexate, a competitive inhibitor of DHFR, can further select for those cells expressing the highest levels of DHFR, and thus, select for the top recombinant protein producers.
Interactions
Dihydrofolate reductase has been shown to interact with
GroEL
GroEL is a protein which belongs to the chaperonin family of Chaperone (protein), molecular chaperones, and is found in many bacteria. It is required for the proper protein folding, folding of many proteins. To function properly, GroEL requires ...
and
Mdm2.
Interactive pathway map
References
Further reading
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
External links
1988 Nobel lecture in MedicineProteopedia: ''Dihydrofolate reductase''*
{{DEFAULTSORT:Dihydrofolate Reductase
Protein domains
EC 1.5.1
Enzymes of known structure